This invention primarily relates to the method and use of fractions isolated or derived from hops as inhibitors of COX-2 and/or 5-LOX activity, particularly reduced isoalpha acids (RIAA), isoalpha acids (IAA), tetrahydroisoalpha acids (THIAA), hexahydroisoalpha acids (HHIAA), alpha acids, beta acids, spent hops, and hop essential oils.
Thrombosis—the current most common cause of ischaemic cardiovascular disease (CVD) such as myocardial infarction and stroke—is the late complication of atherosclerosis, a progressive inflammatory disease characterized by lipid infiltration in the wall of large arteries (atherosclerotic plaques). Platelet and leukocyte recruitment on endothelial cells constitutes an early mechanism of vascular inflammatory damage and consequent vessel occlusion. The increasing appreciation of the role of inflammation in atherosclerosis and thrombosis has renewed interest in the possibility that anti-inflammatory compounds might be effective in the prevention of CVD. Such an intriguing possibility was first raised when acetylsalicylic acid (aspirin) was shown to reduce platelet aggregation induced by several physiological stimuli. Because platelet aggregation was known to play a crucial role in thrombosis, it was anticipated that the newly described anti-aggregating activity of aspirin (at that time, a 70-year-old anti-inflammatory drug) might translate to a clinical benefit in CVD. Aspirin was then tested in dozens of clinical trials and was shown to reduce, by approximately 25%, both primary and secondary incidence of myocardial infarction and other CVDs. However, the gastric side-effects (mainly haemorrhagic) associated with aspirin limited its widespread clinical use for the prevention of cardiovascular events.
The anti-thrombotic effect of aspirin had been related to the inhibition of the platelet enzyme cyclooxygenase (COX), which catalyses the first step in the formation of thromboxane A2 (TxA2), an arachidonic-acid-derived prostanoid that initiates platelet aggregation. Because inhibition of COX in the gastric mucosa would also prevent the formation of cytoprotective prostaglandins, the beneficial anti-platelet effect of aspirin appeared to be inseparable from its gastric side-effects. Following the discovery of a second isoform of the COX enzyme in leukocytes and inflamed tissues, the constitutive enzyme, already described in platelets and endothelial cells, was named COX-1 to distinguish it from the inducible, comparatively aspirin-insensitive COX-2.
Among several alternatives to aspirin, such as the use of drugs that inhibit platelet function without affecting COX-1 activity, two relatively newer antithrombotic approaches will be discussed, both of which are based on the modulation of arachidonic acid metabolism in cells other than platelets, such as blood leukocytes. The first approach focuses on the development of selective drugs that inhibit the production of inflammatory prostaglandins catalyzed by COX-2. This approach leaves COX-1-dependent gastric mucosal function intact but does not prevent platelet activation. Although variable and transient expression of COX-2 has been reported recently in platelets in different clinical settings, it is unlikely that COX-2 inhibitors would affect TxA2-dependent platelet function. The second approach considers 5-lipoxygenase (5-LOX), an enzyme that catalyses the formation of leukotrienes (LTs), as a novel potential target to reduce the atherogenic and thrombogenic role of leukocytes and platelets and their interaction.
Forty years ago, activated platelets were shown to produce inflammatory prostaglandins (PGE2 and PGF2α), a reaction prevented by aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs). In the following few years, arachidonic acid, the 20-carbon-atom fatty acid precursor of prostaglandins, was identified as an initiator of platelet aggregation following its rapid metabolism to intermediate prostaglandin endoperoxides and TxA2, the labile end-products in platelets. The long-lasting (several days) activity of aspirin was attributed to its acetyl group, which was indeed able to irreversibly inactivate COX-1, the platelet enzyme that catalyses the first steps of the arachidonic acid metabolism cascade.
Prostacyclin (PGI2), another metabolite of arachidonic acid produced by the action of COX-1, has been identified in endothelial cells. Because PGI2, in contrast to TxA2, inhibits platelet aggregation, doubts were raised about the clinical potential of aspirin as an anti-thrombotic drug. The assumption was made that to achieve full anti-thrombotic efficacy, the inhibitory effect of aspirin on platelet COX-1 should be retained while that on vascular COX-1 should be minimized (the so-called ‘aspirin dilemma’). Low-dose aspirin (75-100 mg, daily, p.o., in healthy volunteers), which is virtually devoid of a measurable anti-inflammatory effect, was shown to abolish platelet TxA2 generation while leaving vascular PGI2 formation almost intact. However, the epidemiological observation that any dose of aspirin tested (between 30 and 1500 mg, daily, including the highest doses that inhibit both TxA2 and PGI2 generation) was equally effective as an anti-thrombotic, led many researchers to believe that inhibition of platelet COX-1 was indeed the crucial target of aspirin, with concomitant vascular COX-1 suppression having minor, if any, clinical relevance.
Prostaglandins formed by inducible COX-2, including PGI2 in endothelial cells, mediate the development of classical signs of inflammation, such as leukocyte activation, vasodilatation, pain and edema. Although recent work has shown that both COX-1 and COX-2 expression is increased in response to inflammatory stimuli and COX-2 is constitutive in several tissues, the differences between the two COX isoenzymes have led to the development of new concepts in inflammation and its control.
COX-2 is expressed largely in circulating blood leukocytes, vascular cells and macrophages that infiltrate atherosclerotic plaques. This is consistent with a possible direct contribution of leukocytes to vascular disease and thrombus formation. A possible platelet-mediated thrombogenic role of blood leukocytes is also attracting much attention. Activated platelets can recruit leukocytes at the site of vascular injury and form stable conjugates through an adhesion cascade between platelet P-selectin and the leukocyte β2 integrin Mac-1; conversely, activated leukocytes release substances that activate platelets and degrade the endothelial barrier function. Activated platelets can substitute for endothelial cells in the recruitment and migration of leukocytes through the damaged vessel, and leukocytes can accumulate in a growing thrombus and contribute to further platelet activation and deposition and the initiation of blood clotting.
Activated leukocytes, platelet-neutrophil or platelet-monocyte conjugates have been observed in the peripheral blood of patients with unstable angina. In this clinical condition, the presence of platelet-neutrophil conjugates following coronary angioplasty is a predictive index of acute re-occlusion, whereas in acute myocardial infarction the presence of circulating platelet-monocyte aggregates is a sensitive marker of in vivo platelet activation.
Thus, the intriguing possibility is emerging that at the site of an unstable atherosclerotic plaque platelets might be the initial or amplifying trigger of a localized leukocyte-dependent inflammatory response. This is supported by in vivo studies showing co-localization of platelets and leukocytes within atherosclerotic lesions or in areas of ischaemia-reperfusion injury, and activation of neutrophils across the coronary vascular bed in patients with unstable angina, but not in those with stable angina. Activation of neutrophils might be either a marker or a cause (or both) of a widespread inflammatory process occurring in the coronary vasculature. Depending on the intensity of the inflammatory stimuli, such a process might lead to vasoconstriction and thrombosis.
Formation of platelet-leukocyte aggregates within regions of injured vasculature is accompanied by chemokine synthesis in monocytes and the induction of a respiratory burst in neutrophils. If these new perspectives in the pathophysiology of vascular inflammation and thrombosis are confirmed, the use of COX-2 inhibitors to downregulate leukocyte function and their interaction with platelets should be considered. Indeed, aspirin failed, at least in vitro, to modulate platelet-leukocyte interactions.
Selective COX-2 inhibitors might slow down the progression of atherosclerosis and enhance plaque stability, with a possible decrease in atherothrombotic complications. Low-density lipoprotein (LDL)-receptor-deficient mice, fed a lipid-enriched atherosclerotic diet, develop early atherosclerotic lesions in which COX-2 expression can be detected. In this model, either selective inhibition of COX-2 by rofecoxib or suppression of the gene encoding COX-2 resulted in the prevention of atherosclerotic lesion formation without any modification of serum lipids. Furthermore, in a mouse model of acute myocardial infarction, rofecoxib reduced macrophage infiltration. More recently, celecoxib, another COX-2 inhibitor, was shown to improve endothelial function in patients with coronary artery disease.
Together, these data suggest that COX-2 inhibitors might reduce the inflammatory contribution to vascular damage and atherothrombosis, and have the potential advantage over aspirin of minimal gastric side-effects. Furthermore, an intact platelet function in the presence of COX-2 inhibitors might reduce bleeding complications, which are associated with aspirin treatment. COX-2 inhibition would be particularly beneficial in those patients with arthritis or other chronic inflammatory diseases, who have additional cardiovascular risk.
Unlike COX-2, which is expressed in different cell types, including leukocytes and endothelial cells, another enzyme that catalyses arachidonic acid metabolism, 5-lipoxygenase (5-LOX), is only expressed in a limited number of cells (mostly leukocytes). The metabolic products of arachidonic acid that result from the catalytic activity of 5-LOX are the leukotrienes (LTs), which possess potent pro-inflammatory activities and thus might be involved in CVD. Indeed, some LTs are potent vasoconstrictors and increase coronary vascular resistance. A decrease in the production of LTs in leukocytes by 5-LOX inhibitors might achieve downregulation of leukocyte function without undesired effects on other cells, such as endothelial cells. In this way, the balance between the beneficial and detrimental effects of COX-2 inhibitors possibly linked to the concomitant reduction of pro-inflammatory and anti-inflammatory prostanoids in leukocytes and endothelial cells, respectively, could be overcome.
The transfer of the unstable LTA4 (formed by 5-LOX) from neutrophils to cells that possess LTC4 synthase activity, such as platelets and endothelial cells, gives rise to a process of arachidonic acid transcellular metabolism leading to LTC4 generation. Increased levels of LTC4 have been described in plasma from patients with cerebral infarction, whereas increased urinary excretion of LTE4, a metabolite of LTC4, has been reported following episodes of unstable angina and acute myocardial infarction.
Although the role of neutrophils in inflammation has been linked mainly to the formation of LTB4 (a compound with potent chemo-attractant activities), LTA4 might represent the main metabolite released by neutrophils following 5-LOX activation. Formation of cysteinyl-LTs by cell-cell interaction would then cause coronary vasoconstriction. Thus, inhibition of neutrophil function could not only suppress the direct contribution of these cells to inflammation, but also downregulate the contribution of platelets and other interacting cells.
Cysteinyl-LTs are also thought to be involved in damage to gastric mucosa, a finding that is consistent with some experimental evidence that leukocyte-endothelial cell interaction is a prerequisite for aspirin-induced gastropathy. A reduction in NSAID-induced gastric damage was found in neutropenic rats or by treatment of rabbits with monoclonal antibodies against leukocyte adhesive molecules. In addition, decreased synthesis of cysteinyl-LTs was found in a model of neutrophil-perfused rabbit heart by the use of the monoclonal anti-CD18 antibody. Thus, aspirin might induce the expression of adhesion molecules, such as intercellular cell adhesion molecule 1 (ICAM-1) and P-selectin, on gastric endothelium, which results in leukocyte recruitment and cysteinyl-LT biosynthesis, followed by gastric inflammation and bleeding. The latter consequences could be prevented by inhibition of 5-LOX. However, the precise mechanism(s) underlying this suggestive, but still unproven, sequence of events remain to be elucidated.
Together, the data discussed above suggest that a promising pharmacological approach to reduce cardiovascular events at least as effectively as aspirin but without its gastric side-effects should include the following effects: (1) inhibition of COX-1 to prevent platelet TxA2 formation; (2) inhibition of COX-2 to downregulate leukocyte activation and widespread vascular inflammation; and (3) inhibition of 5-LOX to further, and specifically, reduce leukocyte inflammatory and thrombogenic potential, and to counteract the gastric damage associated with the inhibition of COX-1.
Compounds that are capable of inhibiting both COX and 5-LOX are being developed as anti-inflammatory agents. Further development of some dual inhibitors, including tepoxalin, tebufelone and CI986, has been limited by drug metabolism issues; other dual inhibitors have been shown to have anti-platelet, anti-leukocyte and anti-inflammatory properties together with an improved gastric tolerability.
In particular, licofelone is an effective inhibitor of both TxA2-mediated platelet function and neutrophil activation, the latter measured as LTB4 formation, generation of reactive oxygen species, elastase release and homotypic aggregation induced by different inflammatory stimuli. Licofelone was also able to reduce neutrophil surface expression of Mac-1 and the consequent platelet-neutrophil conjugate formation and transcellular synthesis of LTC4.
In the prevention of atherothrombotic events, licofelone and similar drugs might therefore be at least as effective as low-dose aspirin, without the gastric side-effects of the latter or the pro-thrombotic risk associated with selective COX-2 inhibitors. These drugs might even exert a more powerful anti-thrombotic effect than aspirin through additional anti-inflammatory mechanisms by inhibiting, for example, the availability of intravascular tissue factor from monocytes or endothelial cells exposed to inflammatory agonists, or to prevent tissue factor transfer from leukocytes to platelets. The potential downregulation of blood clotting by dual inhibitors, however, remains to be defined and is the subject of active investigation. In this context, recent reports that NO-aspirin (NCX4016) reduces 5-LOX activity and blunts monocyte tissue factor expression are of interest.
A promising anti-thrombotic approach to minimize the gastric side-effects of aspirin and the cardiovascular risk of COX-2 inhibitors is to concurrently depress the activities of both COX and 5-LOX enzymes. Several such dual inhibitors of the production of PGs and LTs in vitro have been identified. Some of these inhibitors, such as licofelone, are presently being evaluated in Phase III clinical studies for the treatment of osteoarthritis. The translation of the promising preclinical and safety profile to the clinical arena awaits the completion of thorough clinical investigations.
Thus, only large-scale randomized controlled clinical trials will show whether, while representing a better and/or safer tool for the treatment of inflammatory disorders, these new drugs can also antagonize interactions between blood and vascular cells that promote inflammatory events, such as atherothrombosis, thus reducing the risk of clinical cardiovascular outcomes.
5-LOX has also been implicated in the progression of certain cancers. For example, see Ding, et. al., Biochem. Biophy. Res. Comm., 261, 218-223 (1999) and Shureiqi, et. al., Cancer Res., 61, 6307-6312 (2001). For a detailed discussion of the advantages of dual COX-2/5-LOX inhibitors see, for example, Charlier, et. al., Eur. J. Med. Chem., 38, 645-659 (2003).
We have surprisingly discovered that fractions derived or isolated from hops have utility in treating the myriad of diseases associated with hyperactivity of COX-2 and/or 5-LOX.